Human ferredoxin belongs to the vertebrate ferredoxin family, which includes bovine adrenodoxin. It is a small (13.8 kDa) acidic protein with a [2Fe-2S] cluster. It functions as an electron shuttle in the cholesterol side-chain cleavage reaction, which is the first step of steroid hormone biosynthesis. The protein studied here was produced in Escherichia coli and doubly labeled with 13C and 15N. The diamagnetic 15N, 13C?, 13C?, 13C?, 1H? and 1HN resonances from about 70% of the 124 amino acid residues for oxidized human ferredoxin and 80% of those for the reduced protein have been assigned primarily on the basis of results from three-dimensional, triple-resonance experiments. Secondary structure features for oxidized protein have been identified from a combination of the chemical shift index and sequential NOE data, and those for the reduced protein have been deduced from chemical shift index analysis. Comparison of structural information on the oxidized and reduced proteins reveals that a structural change accompanies the reduction of the [2Fe-2S] cluster. Major structural changes are localized at two regions: residues 29?31 and residues 109?124, which form part of the C-terminal region of the protein. The possible functional significance of these oxidation-state-dependent structural changes is discussed.